NF‐Y‐dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage

Abstract Glutamate receptor subunit 4 (GluA4) is highly expressed by neural cells sensitive to excitotoxicity, and is the predominant subunit expressed by oligodendrocyte precursor cells (OPC) during a key period of vulnerability to hypoxic‐ischemic injury. Therefore, transcriptional networks downstream of excitotoxic GluA4 activation represent a promising area for therapeutic intervention. In this work, we identify the CCAAT binding transcription factor NF‐Yb as a novel transcriptional regulator of Gria4 (GluA4 gene), and a controller of excitotoxic death in the oligodendroglial lineage. We describe a novel regulatory region within Gria4 containing CCAAT sequences whose binding by NF‐Yb is regulated by excitotoxicity. Excitotoxicity‐induced alterations in NF‐Yb binding are associated with changes in Gria4 transcription, while knockdown of NF‐Yb alters the transcription of reporter constructs containing this regulatory region. Data from immortalized and primary OPC reveal that RNAi and pharmacological disruption of NF‐Yb alter Gria4 transcription, with the latter inducing apoptosis and influencing a set of apoptotic genes similarly regulated during excitotoxicity. These data provide the first definition of a trans‐acting mechanism regulating Gria4, and identify the NF‐Y network as a potential source of pharmacological targets for promoting OPC survival.

Excitotoxic injury induces OPC and oligodendrocyte cell death through stress-induced apoptotic pathways involving the Bcl-2 family (Ness, Romanko, Rothstein, Wood, & Levison, 2001;Ness, Scaduto, & Wood, 2004;Sanchez-Gomez, Alberdi, Ibarretxe, Torre, & Matute, 2003;Sanchez-Gomez, Alberdi, Perez-Navarro, Alberch, & Matute, 2011;Simonishvili, Jain, Li, Levison, & Wood, 2013). These processes are tightly regulated by the expression of pro-and anti-apoptotic Bcl-2 genes (Kumar & Cakouros, 2004;Riley, Sontag, Chen, & Levine, 2008), thus the transcriptional networks stimulated by excitotoxic injury represent promising targets for therapies aiming to reduce excitotoxic injury and cell death. In the context of OPC the transcriptional events associated with GluA4 are of particular interest due to its prominent expression in these cells, and its links to the induction of excitotoxic cell death (Page & Everitt, 1995;Santos et al., 2006). Based on this premise we used an excitotoxic injury model in the Oli-neu cell line (Jung et al., 1995) and primary OPC (pOPC) to identify subunit B of the nuclear factor Y complex (NF-Yb) as a regulator of GluA4 transcription and cell survival in oligodendroglia. Using a combination of ChiP, qPCR, Western blot and reporter assays we show that excitotoxic AMPAR stimulation alters NF-Yb binding to a novel Gria4 regulatory region, leading to complementary alterations in the levels of GluA4 mRNA and protein. We also provide data highlighting the therapeutic potential of the NF-Y transcriptome, with siRNA and pharmacological-mediated disruption of the NFY pathway compromising oligodendroglial viability and regulating similar apoptotic genes to those influenced by excitotoxic injury.
Garcinol was used at 10 mM for gene expression, Western blot, and ChIP experiments. This concentration was selected based on pilot studies examining treatment with log concentrations of Garcinol (1-1,000 mM), in which concentrations >10 mM caused catastrophic cell detachment and death (data not shown). BrdU (Sigma-Aldrich Company Ltd) stocks were prepared as described by Fannon et al. (2015).

| Trypan blue cell viability assay
For viability assays Oli-neu were seeded into 6-well plates at a density of 5 3 10 5 cells/well, while pOPC were cultured in 12-well plates at a density of 5 3 10 4 cells/well. 24 hr after seeding cells were treated with L-glutamate, CTZ, or AMPA/CTZ for 5 hr, then trypsinized, stained with 0.04% trypan blue (Sigma-Aldrich) and counted on a haemocytometer to quantify % viability. Data were collected in triplicate from five independent cell cultures by an experimenter masked to the treatment.

| Antibodies
Antibodies used in this study are detailed in Supporting Information Table S1. Specificity for immunopositive signals was determined in control samples incubated with secondary antibodies alone.
2.5 | Immunocytochemistry, plasma membrane labelling and imaging Immunofluorescent staining was performed using a protocol previously used in our lab to label brain slices (Fannon et al., 2015). The protocol was adapted for use on Oli-neu cells as follows. Oli-neu were grown in 8-well chamber slides (Corning Falcon, Wiesbaden, Germany) at a density of 3 3 10 4 cells/well, and the fixation, blocking, and secondary antibody incubation steps were reduced to 20 min, 1 hr, and 1 hr, respectively. Chamber slides were coverslipped with Vectashield containing DAPI (Vector Laboratories, Peterborough, UK). CellMask Deep Red (Molecular Probes, Eugene, OR; Thermo Fisher Scientific, Waltham, MA) was used to localize GluA4 to plasma membranes of Olineu cells. Live cells were incubated in CellMask (2.5 ml/ml) diluted in Sato medium for 5 min (5% CO 2, 378C) and immediately fixed in PFA, permeabalized with an optimized protocol (0.2% Tween-20 for 10 min) which preserved CellMask signal while allowing reasonable labeling of GluA4. GluA4 was then detected as described above, except for omission of Triton-X100 in the blocking and carrier solutions. Slides were imaged on an inverted microscope (Zeiss Axiovert 200, Zeiss, Oberkochen, Germany) equipped with a differential spinning disk confocal module as described previously (Fannon et al., 2015).Objectives used were 203 (Air, 0.5 N.A.) and 1003 (oil, 1.3 N.A.). AMPAR immunofluorescence was imaged in confocal mode, while proliferation studies were imaged in the wide-field configuration. Cleaved caspase-3 labeling was imaged at 203 on Axioplan 200 epi-fluorescent microscope equipped with an AxioCam HRc (all from Zeiss, Herefordshire, UK).

| Proliferation and apoptosis studies
Cells were seeded into 8-well chamber slides at a density of 3 3 10 5 and allowed to settle for 24 hr prior to treatment with AMPA/CTZ, followed by exposure to BrdU (10 mM) for 7 hr. For AMPA 24h experiments BrdU treatments were introduced during the last 7 hr of treatment. BrdU was detected as previously described (Fannon et al., 2015) and proliferation quantified as the % DAPI 1 /BrdU 1 cells from five randomly selected fields/well from seven to eight independent well replicates. Apoptosis was examined by immunofluorescent labelling for cleaved Caspase3 24 hr after treatment of cells with Garcinol (5 hr, 10 mM), or the vehicle control (DMSO, 0.001%). Data were quantified as % cleaved caspase-3 1 /DAPI 1 nuclei from four randomly selected areas of each well (n 5 4/group).

| RNA and protein extraction
Five independent cultures of Oli-neu cells were seeded into T25 (1 3 10 6 cells/flask) in duplicate and left to adhere overnight. Duplicate flasks received the following treatment pairings: CTZ alone or AMPA/ CTZ; Control Sato or AMPA 24h ; DMSO control or Garcinol (see Section 2.2 for treatment durations). Trypsinized samples were divided, washed with ice cold PBS, and RNA extracted from one sample using the RNeasy mini kit (Qiagen, Manchester, UK).The other sample was incubated for 30 min on ice with protein lysis buffer consisting of 20 nM Tris HCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Igepal and 5 ml/ml of protease inhibitor. Protein levels were then quantified using the RC/DC assay (Bio-Rad, Hemel Hempstead, UK) and RNA/ protein samples were stored at 2708C pending further analysis. RNA extractions from pOPC were performed using an identical protocol.

| Gene expression analysis and NF-yb knockdown
RNA samples were reverse transcribed using the Tetro cDNA synthesis kit (Bioline Reagents Ltd, London, UK) using the random hexamer primers provided. qPCR was performed using a Bio-rad iQ5 PCR detection system with SYBR green PCR mastermix (Thermo Fisher Scientific, Paisley, UK, see Supporting Information Table S2 for qPCR primer sequences). Primer efficiency was tested using a 7-point standard curve, and gene expression calculated using the 2 -DDCt method and were mixed with FuGENE HD (Promega Corporation, Madison, WI) at a ratio of 1:4 (mg cDNA:ml reagent), and cells transfected with the resulting complexes. NF-Yb gene expression was examined 24 and 48 hr after transfection. In experiment using pOPC, siRNA constructs (40 nM) were mixed with Lipofectamine RNAiMAX (Thermo fisher Scientific). pOPC were incubated in the Liposome-siRNA complexes for 6 hr, after which they were washed with OPC medium, and then cultured for a further 24 hr prior to use in experiments.

| Western blot
Extracted proteins were separated by SDS-Page using the Xcell sure lock mini-cell system (Thermo Fisher Scientific), and then transferred onto PVDF membranes (Immobilion-P, Merck Millipore, Watford, UK).
Membranes were then incubated for 1 hr at room temperature in blocking medium (5% non-fat milk dissolved in TBST [0.05 M Tris, 0.15 M NaCl, pH 7.2, 0.1% (v/v) Tween20]), before incubation in primary antibodies overnight at 48C with constant agitation. After washing in TBST membranes were incubated in secondary antibodies for 1 hr at room temperature. All antibodies were diluted in blocking medium (see Supporting Information Table S1 for details of antibodies).

| Chromatin immunoprecipitation (ChIP)
ChIP experiments quantifying NF-Yb binding to the Gria4 intronic regulatory region were performed in accordance with previously published methods (Weaver et al., 2004;Zhang et al., 2010). Briefly, Oli-neu cells were plated at a density of 1 3 10 6 cells in T25 flasks and allowed to adhere overnight. Duplicate flasks received the following treatment pairings: CTZ alone or AMPA/CTZ; Control Sato or AMPA 24h ; DMSO control or Garcinol. Following these treatments cells were fixed in 1% PFA at RT for 10 min before the addition of 0.125 M glycine. The medium was then removed and saved and cells washed in ice cold PBS before collection with cell scrapers in Farnham lysis buffer (plus protease inhibitors). The stored cell medium and collected cells were then combined and pelleted at 48C, before sonication (Soniprep 150 plus, MSE, Ltd, London, UK, [1 3 10 s maximum power]), followed by centrifugation of the resulting suspension and further sonication of supernatants at 7 3 10 s/ 20 amp. Supernatants were de-crosslinked and purified to determine consistent fragment sizes on a 1.5% agarose gel. Chromatin was immunoprecipitated with an antibody against NF-Yb (Supporting Information   Table S1) while antibodies against RNA POL and normal mouse IgG were used for positive and negative controls respectively. Samples were then prepared for DNA elution by hydrolysis of crosslinks and all inputdigested DNA and output DNA purified using the Qiaquick PCR purification kit (Qiagen). Ratios of output DNA:input DNA were determined by qPCR with the primers detailed in Supporting Information

| Transcriptome analysis
Transcriptomic profiling was carried out on extracted RNA (Qiagen, Crawley, UK) after the quality of RNA was assessed using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE) and qualified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Agilent two color 60K Mouse microarrays were used to assess whole genome gene expression according to the manufacturer's instructions.
Control samples were labelled with Cy3 (green) and treated samples were labelled with Cy5 (red). For background correction, the linear models for microarray data (LIMMA) package (Ritchie et al., 2015) were used within R 3.3.2 (RCoreTeam, 2016). Normalization within arrays was conducted using the "Loess" function and normalization between arrays was conducted using the "Aquantile" function (Smyth & Speed, 2003). The "lmscFit" function was used for separate channel analysis of two-color microarray data (Smyth & Altman, 2013) using Benjamini-Hochberg to adjust p values.

| Analysis of gene expression network models
To derive an interactome model for AMPA/CTZ and Garcinol treated cells, differentially expressed genes were used as "seeds" and all known protein/protein interactions between the seeds and their inferred immediate neighbors were identified to generate a biological network using the BioGRID model of the human Interactome String database was used to assess the integrity and connectivity of gene modules (Szklarczyk et al., 2015).

| Data analysis and statistics
Normal distributions were tested using an online Shapiro-Wilks tool (Dittami, 2009) and when present two-group comparisons were made by t tests, and multiple comparisons examined by one-way ANOVA followed by Tukey's Honest Significance Difference post hoc tests.
Mann-Whitney U tests were used for two-group comparisons when non-normal distributions were detected. All comparisons were tested using the online tool VassarStats (Lowry, 2009). Potential regulators of Gria4 were identified by screening all known Gria4 interactions (Ingenuity Pathway Analysis, Qiagen, Redwood City, CA). A fixed value of p a < .05 for two-tailed tests was the criterion for reliable differences between groups. One-tailed tests were used when predictions were made possible by preceding results. Cited values are s 6 SEMs.
AMPAR expression has not been reported in Oli-neu cells so we used qPCR and immunocytochemistry to detect AMPAR subunits GluA1-4. qPCR detected transcripts for all four GluA subunits (data not shown).
Using immunofluorescent labeling GluA1 expression was limited to a few cells in which expression was restricted to the nucleus (Supporting Information Figure S1ai, S1aii). In contrast, GluA2, 3, and 4 were expressed by all cells (GluA2-3, Supporting Information Figure S1bi  Figure S2a). These data suggest that functional calcium-permeable AMPAR are present at modest levels in Oli-neu cells. We examined the ability of these receptors to mediate pathological signaling events by exposing Oli-neu cells to AMPAR-stimulating treatments likely to induce excitoxicity. Log concentrations of L-glutamate (1-1,000 mM) applied for 5 hr reduced viability from 10 mM (Supporting Information Figure S2b; ANOVA p < .0001, control vs. 10 mM p < .05, control vs. 100 mM p < .01, control vs.
1,000 mM p < .01). We also examined viability after a 5-hr exposure to AMPA/CTZ, which specifically and potently activates AMPAR without affecting other types of glutamate receptor. This treatment was more effective than L-glutamate, with effects observed at the lowest concentration (1 mM; Figure 1e; ANOVA p < .0001, control vs. 1 mM p < .01, control vs. 10 mM p < .01, control vs. 100 mM p < .01, control vs.
Prolonged stimulation with AMPA (200 mm) in the absence of CTZ inhibits OPC proliferation and differentiation without affecting viability (Yuan et al., 1998). We used this treatment, designated here as AMPA 24h, to determine if Oli-neu cells are similarly sensitive to milder levels of AMPAR activation. AMPA 24h had no effect on Oli-neu cell proliferation (Supporting Information Figure S3a; p 5 .83), or cell viability (Supporting Information Figure S3b; p 5 .38), showing that AMPA only affected Oli-neu cells when co-treated with CTZ. Incubation with 100 mm AMPA/CTZ produced a reliable decrease in cell viability ( Figure   1e), so this treatment was chosen for studies aiming to uncover mechanisms regulating GluA4 transcription during excitotoxic injury.

| Identifying transcriptional regulators of Gria4
An in silico approach designed to identify regulators of Gria4 transcription highlighted NF-Yb as a relevant target. Gria4 was previously identified in a large-scale screen of NF-Yb binding sites carried out in HEPG2 cells (Testa et al., 2005). This information, together with its established role in controlling proliferation and cell death (Imbriano, Gnesutta, & Mantovani, 2012), actions that are regulated in oligodendroglia by AMPAR, marked NF-Yb as a promising candidate for further study. The human Gria4 gene was screened for locations showing evidence of NF-Y binding. This search identified a candidate region  Table S2). This region is located 3 0 to the transcription start site (TSS) and so falls outside of the classic proximal promoter position associated with CCAAT elements (Mantovani, 1999).

| Excitotoxic AMPAR stimulation decreased expression of NF-yb and GluA4
Prolonged activation of AMPAR in pOPC cultures has been shown to induce alterations in the expression of GluA4 protein (Hossain et al., 2014). To probe possible mechanisms regulating Gria4 transcription, we measured Gria4 and NF-Yb transcript and protein in Oli-neu cells subjected to AMPA 24h . Similar to our observations of cell viability and proliferation, transcription of Gria4 and NF-Yb remained unaffected by AMPA 24h (Supporting Information Figure S4a,b; Gria4, p 5 .98; NF-Yb, p 5 0.64). Levels of GluA4 (Supporting Information Figure S4ci,cii) and NF-Yb (Supporting Information Figure S4ci,ciii) protein were also unaffected by this AMPA treatment (GluA4, p 5 .75; NF-Yb, p 5 .86).
Given the modest extent of functional AMAPR expression in Oli-neu cells we considered whether stronger AMPAR activation might be required to modulate GluA4 expression. Indeed, treatment of Oli-neu with AMPA/CTZ for 5 hr led to a significant reduction in the level of

| Excitotoxic AMPAR stimulation decreased enrichment of NF-yb at Gria4 binding sites
The corresponding changes in NF-Yb and Gria4 detected after AMPA/CTZ (Figure 2) may indicate that both genes are targets of the same regulator, or that NF-Yb directly regulates Gria4. We performed ChiP assays directed at CCAAT sites 1-3 (Supporting Information Table S3) to determine whether NF-Yb physically interacted BEGUM ET AL. | 5 with these CCAAT sites, and if binding was modulated by excitotoxic injury. Treatment with AMPA/CTZ resulted in a significant reduction in NF-Yb enrichment at sites 1 (p < .0001; Figure 3a) and 2 (p < .01; Figure 3b) but not site 3 (data not shown). In agreement with our analysis of Gria4 transcription (Supporting Information Figure S3), AMPA 24h treatment had no effect on NF-Yb binding (sites 1 and 2; Figure 3c,d; site 3 data not shown; site 1, p 5 .63; site 2, p 5 .31). The observed decrease in Gria4 F IG URE 1 . 6 | transcription following AMPA/CTZ (Figure 2a), and the ChIP data, suggested that NF-Yb-Gria4 interactions play an important role in the regulation of Gria4 transcription.

| Pharmacological inhibition of NF-Y reduced NFyb function and triggers apoptotic cell death
The data presented above indicate NF-Y as a regulator of Gria4 transcription during excitotoxic injury. Further support for this idea was sought by performing pharmacological experiments to disrupt NF-Y activity in Oli-neu cells. NF-Y function depends on interactions between NF-Yb and the nuclear co-activator protein p300 (Figure 4a; Faniello et al., 1999). Therefore, we used the p300 inhibitor Garcinol (Balasubramanyam et al., 2004)  site 2, p < .05), but not 3 (data not shown). These data confirm that Garcinol induced the same changes in NF-Yb/Gria4 interactions, and Gria4 transcription to those observed after AMPAR-mediated excitotoxicity. To determine if disruption of NF-Ywas associated with apoptosis, we examined nuclear labeling for cleaved caspase-3 following a 5-hr Garcinol treatment. 24 hr after this treatment cleaved caspase-3 was undetectable in vehicle treated cells (Figure 4gi,gii), but could be observed in the cytoplasm and nuclei of Oli-neu cells exposed to Garcinol (Figure 4giii,giv). The % of cleaved caspase-3 1 cells was 25% (Figure 4h), suggesting caspase-3-dependent apoptosis.

| NF-yb knockdown reduced transcription of constructs containing the Gria4 regulatory region
To test the influence of NF-Y on the CCAAT-containing regulatory region we performed reporter assays on Oli-neu cells transfected with the wtGria4 construct (Supporting Information Table S3) and either siNF-Yb or siControl. Knockdown of NF-Yb reduced reporter activity from the wtGria4 construct by 54% relative to siControl transfected cells ( Figure 5a; p < .0001). To verify the efficiency of the knockdown, we measured NF-Yb transcripts 24 hr after transfection with siNF-Yb. Olineu cells transfected with siNF-Yb displayed a 64% reduction in NF-Yb mRNA compared with cells transfected with siControl (Figure 5b; p < .01). NF-Yb knockdown also reduced the level of Gria4 transcription ( Figure 5c; p < .05) further supporting a role for NF-Y in the control of Gria4 expression. We sought additional evidence for the involvement of NF-Y in Gria4 transcription by generating a reporter construct containing mutations in NF-Yb binding sites 1-3 (DGria4, Supporting Information Table S3). In contrast to NF-Yb knockdown, cells transfected with DGria4 exhibited a 27.6-fold increase in activity of the reporter compared with cells treated with the wtGluA4 construct (Figure 5d; p < .01).  Table S8). The shared network of 39 genes was more connected than expected by chance (p < .001) and a central element was identified (dashed blue ellipse in Figure 7b,c) in which treatments induced identical differential gene expression.

| DISCUSSION
In this work, we used excitotoxic levels of AMPAR stimulation to uncover mechanisms regulating the expression of GluA4 in cells of the oligodendrocyte lineage. We identified an intronic regulatory region harboring binding sites for NF-Yb, whose binding by this transcription factor are down-regulated during excitotoxic injury. Decreased NF-Yb binding is accompanied by a reduction in the levels of GluA4 mRNA and protein, an effect mimicked at the transcriptional level by siRNA knockdown of NF-Yb. We also show that reducing NF-Y function through pharmacological inhibition and knockdown compromises the survival of Oli-neu and pOPC respectively, and that reduced NF-Y F IG URE 4 . function alters the expression of a number of apoptotic genes.
Together, these data provide the first example of a trans-acting mechanism controlling Gria4 expression, and highlight the NF-Y pathway as a regulator of OPC viability and survival.

| Studying transcriptional regulation of AMPAR in oli-neu cells
Oli-neu cells have been used to study aspects of oligodendrocyte biology including myelin protein trafficking (Dhaunchak, Colman, & Nave, 2011;Trajkovic et al., 2006), exosome release (Fr€ uhbeis et al., 2013), and the expression and function of connexins (Sohl, Hombach, Degen, & Odermatt, 2013) and G protein coupled receptors (Fratangeli et al., 2013;Simon et al., 2016). The ease of generating large, high purity, cultures of these cells, and their ease of transfection relative to pOPC, make them particularly useful for studies of transcript expression and regulation where large numbers of homogenous cells must be manipulated and harvested for mRNA and protein sampling (Gobert et al., 2009;Iacobas & Iacobas, 2010;Joubert et al., 2010). In this regard, their utility as a model for OPC biology is further supported by data from the present study showing that alterations in NF-Y function induce produces similar effects on Gria4 transcription and cell viability in both Olie-neu and pOPC.
The expression and regulation of glutamate receptors has not been explored in Oli-neu cells, although this was examined in electrophysiological studies of the closely related cb-neu cell line (Jung et al., 1995), in which the absence of membrane currents was reported following exogenous application of glutamate. In contrast, the present paper provides two independent lines of evidence (Ca 21 imaging and excitotoxic cell death) that suggest modest levels of functional AMPAR expression in Oli-neu. Despite these low levels of AMPAR function, Oli-neu cells proved useful for detecting excitotoxic changes in GluA4 subunit expression and the transcriptional machinery controlling these processes.

| Excitotoxicity-induced changes in Gria4 transcription in olie-neu and pOPC
Depending on the cell type examined, excitotoxic injury either decreased (Oli-neu) or increased (pOPC) transcription of Gria4. These data from pOPC reflect those from an in vivo study of GluA4 expression in neonatal rats exposed to hypoxia (Sivakumar, Ling, Lu, & Kaur, 2010). Here, exposure to hypoxia that induces strong elevations in the concentration of extracellular glutamate is associated with a significant increase in GluA4 immunoreactivity within immature oligodendroglia.
The expression of Gria4 is regulated developmentally (Itoh et al., 2002;Hossain et al., 2014), thus the direction of excitotoxicity-induced regulation of Gria4 in oligodendroglia may depend on maturational status.
Indeed, Oli-neu cells cultured under the conditions used in the present study exhibit low levels of immunoreactivity to anti-O4 (Toutouna et al., 2016), suggesting that this cell line holds a more immature position in the lineage than pOPC, which typically exhibit strong anti-04 staining. This difference not withstanding, other aspects of the relationship between NF-Yb, Gria4 transcription and cell viability were validated in pOPC, supporting a role for NF-Yb in the regulation of Gria4 and cell survival in OPC.

| Characteristics of the intronic CCAAT regulatory region in mouse Gria4
The CCAAT box is a common promoter element occupying a position typically located between 260 and 2100 from the TSS (Mantovani, 1999), where it exerts well-described actions on promoter activity (Dolfini, Gatta, & Mantovani, 2012). Prior to the present work NF-Y had not been studied in oligodendroglia. However, CCAAT sites located in the promoter of protein zero, a key myelin gene in the peripheral nervous system, regulate protein zero transcription in Schwann cells (Brown & Lemke, 1997). The CCAAT sites identified in the present paper are located 3 0 to the Gria4 TSS so fall outside of the proximal promoter region. These observations add to a growing body of evidence demonstrating roles for NF-Y outside of classic promoter regions. For example, a recent genome-wide mapping study revealed that 25% of NF-Y peaks are associated with distal enhancer regions (Fleming et al., 2013).
In addition, expression arrays performed following NF-Ya inactivation revealed that 50% of differentially expressed genes exhibit NF-Y/DNA binding outside of promoter regions. The importance of NF-Y/ enhancer interactions have also been validated in vivo, where they are found to regulate tissue specific expression of Hoxb4 during embryonic development (Gilthorpe et al., 2002).  (Faniello et al., 1999). (b) Garcinol reduces Olineu cell viability. Cell viability normalized to the control treatment was reduced following a 5-hr treatment with Garcinol (10 mM; control 1.00 6 0.02, Garcinol 0.88 6 0.01). (c) Transcript levels for NF-Yb were significantly reduced by a 5-hr exposure to Garcinol (control 1.00 6 0.15, Garcinol 0.55 6 0.06). (d) Garcinol reduced levels of GluA4 transcripts (control 1.00 6 0.19, Garcinol 0.51 6 0.06). (e and f) Treatment with Garcinol disrupted binding of NF-Y to the Gria4 promoter. Top panels show RT-PCR gels for four independent experiments on sites 1 (e) and 2 (f). I, input signal; O, output signal; Con, control; G, Garcinol. Lower panels show quantification of NF-Yb enrichment at sites 1(e) and 2 (e). Enrichment of NF-Yb at both sites was significantly reduced following treatment with Garcinol at site 1 (control 1.00 6 0.25, Garcinol 0.16 6 0.08) and site 2 (control 1.00 6 0.19, Garcinol 0.25 6 0.12). (g) Representative cleaved Caspase-3 1 immunostaining (green) with DAPI (blue) in Oli-neu cells after 24-hr treatment with DMSO vehicle (gi and gii) and Garcinol (giii and giv). White arrowheads indicate locations with cleaved Caspase3 1 immunofluorescence within the nucleus indicative of apoptosis. (h) Cleaved caspase-3 1 was undetectable in control treated cells, but was observed in 25% of cells after Garcinol treatment. * and ** Significance p < .05 and p < .01 respectively, # significance p < . Knockdown of NF-Yb reduces pOPC cell viability. pOPC viability normalized to siControl values was reduced 24 hr after transfection with siNF-Yb multi (siControl 100 6 2.73, siNF-Yb multi 84.99 6 5.01). (c) Excitotoxic treatment compromises the viability of pOPC. The proportion of viable pOPC (normalized to CTZ control) is significantly reduced after 5 hr of AMPA/CTZ (CTZ control 100 6 2.00, AMPA/CTZ 78.25 6 2.33). (di and dii) Excitotoxic stimulation increases transcription of NF-Yb and Gria4 relative to CTZ control levels (NF-Yb: CTZ control 1.00 6 0.01, AMPA/CTZ 1.57 6 0.10; Gria4: CTZ control 1.03 6 0.10, AMPA/CTZ 2.25 6 0.17). *, **, ** Significance p < .05, p < .01, and p < .001, respectively. Data are expressed as means 6 SEM Excitotoxic injury reduced NF-Yb binding to CCAAT sites located within the Gria4 intronic regulatory region and produced a parallel reduction in the level of Gria4 transcripts. In agreement with this, our functional studies using a reporter construct containing the Gria4 regulatory regions showed that knockdown of NF-Yb reduced activity of a minimal promoter. Overall these data from qPCR, ChiP and reporter assays are consistent with a role for NF-Y in regulating Gria4 transcription in Oli-neu cells. We also examined NF-Y actions at these sites using a reporter construct bearing mutations designed to prevent NF-Y binding. Surprisingly, these mutations led to a significant increase in reporter activity. Knockdown of NF-Yb had no effect on promoter activity when the Gria4 region was absent. Thus, the opposing actions of the NF-Y site mutations and siRNA knockdown, cannot be explained on the basis of NF-Y actions on sequences located outside of the regulatory region. These results could be explained if mutation of the binding sites hindered the actions of another repressive trans-acting factor that exerts actions on the promoter via the CCAAT elements. Indeed, the high abundance of H3K27Ac marks in this region of Gria4 suggests it is highly regulated. One possible candidate is the CCAAT/enhancer binding proteins (C/EBPs), which have a well described ability to bind CCAAT boxes (Landschulz, Johnson, Adashi, Graves, & McKnight, 1988). Irrespective of these possibilities, the knockdown, qPCR and ChIP experiments presented in this paper highlight NF-Yb as an effective regulator of Gria4 transcription.
4.4 | First trans-acting mechanism regulating GluA4: Implications for protective therapies To our knowledge, the actions of NF-Yb reported in the present study describe the first trans-acting mechanism controlling GluA4 expression.
Earlier work examining the promoter of rat Gria4 described two potential regulatory regions (Borges, Myers, Zhang, & Dingledine, 2003). The first was a long interspersed element (LINE) located 3.6-3.9 kb upstream from the translation start site (TSS), which was found to contain silencing elements. This work also identified several TSS in Rat Gria4 located between 21,090 and 21,011. Importantly, the LINE silencing region is distant from these sites being 2-3 kb further upstream, and so contrasts greatly with the regulatory region we have described in mouse Gria4, which is located 3 0 of the TSS. The second regulatory region in Rat Gria4 contained a cluster of transcription factor sites (e.g., SP1, CAAT box, IK2, MRE) located close to the TSS (Borges et al., 2003). Although the authors did not determine the influence of these sites on transcription directly, they suggested their involvement in directing neuronal selectivity since deletion of the 177 bp region containing them reduced transcription of Gria4 in neuronal, but not glial cultures.
New data from the present study indicate that NF-Y plays an important role in the response of oligodendroglia to cellular stress.
Both excitotoxic injury and a pro-apoptotic treatment with Garcinol initiated a change in NF-Y function that was tightly linked to alterations in Gria4 transcription. Reduced levels of GluA4 could protect oligodendrocytes from further injury by diminishing the supply of calciumpermeable subunits for assembly into AMPAR. In contrast, recent FIG URE 7 Interactome modelling from AMPA/CTZ and Garcinol treatments identified a core network element with consistent differential gene expression. (a) Overlap of the central network hierarchies identified by the moduland algorithm (numbers of genes). A network of the common 39 differentially regulated genes was defined using the STRING database (confidence > 0.4) in Oli-neu cells treated with AMPA/CTZ (b) and Garcinol (c). Blue 5 inferred interaction; green 5 down regulated; red 5 up regulated gene expression with Oli-neu cell treatments. Shade of green/red indicate level of fold change. Rounded squares 5 genes associated with control of apoptosis. All differential gene expression p < .01 [Color figure can be viewed at wileyonlinelibrary.com] findings indicating a role for AMPAR subunits GluA2, 3, and 4 in OPC survival (Kougioumtzidou et al., 2017) suggest that an increase in Gria4 transcription, as observed in pOPC following excitotoxic treatment, may promote viability. Given this complexity, further studies are required to determine the potential of Gria4 modulation to promote OPC viability under excitotoxicity-inducing conditions such as hypoxicischemia (Follett et al., 2000) and CNS inflammation (Groom et al., 2003;Kanwar et al., 2004;Pitt et al., 2000).

| Involvement of NF-Y in cell death
Both excitotoxic injury and pharmacological inhibition of NF-Y reduced the function of this transcription factor and compromised viability in Oli-neu cells, while siRNA-mediated knockdown of NF-Yb similarly reduced viability in cultures of pOPC. Disruption of NF-Y function also induced apoptosis and regulated a core group of apoptotic genes that were similarly regulated by excitotoxicity. Together these findings suggest a link between NF-Y, oligodendroglial survival, and the regulation of genes controlling apoptosis. In agreement with this, a number of reports provide evidence for the involvement of NF-Yb in antiapoptotic actions. For example, NF-Yb knockdown in a colorectal carcinoma cell line (HCT116) induces apoptosis through reductions in the anti-apoptotic genes Bcl-2 and BI-1 (Benatti et al., 2008). ChIP assays and luciferase reporter experiments show that Bcl-2 and BI-1 are targets of NF-Yb and that reduced NF-Y function decreases transcription of these genes, indicating that NF-Yb directs the activation of these anti-apoptotic genes (Benatti et al., 2008). In the same study it was reported that reduction of NF-Yb leads to increased acetylation of p53, a condition that promotes apoptosis via increased p53 binding to proapoptotic targets Bax and Mdm2. Thus, NF-Yb may regulate the apoptotic response of cells by controlling the balance between pro-and anti-apoptotic pathways. In line with this idea, recent work in a human osteosarcoma cell line (U2OS) identified NF-Yb as a direct target of pro-apoptotic E2F1 (Jiang, Nevins, Shats, & Chi, 2015). Importantly, knockdown of NF-Yb enhances E2F1-mediated activation of proapoptotic targets and increases apoptosis.
Increasing expression of NF-Yb may not promote survival under all conditions of cellular stress. For example, the pro-apoptotic FAS/APO1 gene is a direct NF-Y target, whose expression in a breast cancer cell line (MCF7) is enhanced by both apoptotic DNA damage, and the forced expression of NF-Y proteins (Morachis, Murawsky, & Emerson, 2010). Similarly, forced transduction of carcinoma cell line NT2/D1 with NF-Ya proteins reduces cellular growth by inducing a decrease in their survival (Mojsin, Topalovic, Marjanovic Vicentic, & Stevanovic, 2015). The relationship between NF-Y function and cell viability therefore appears to be complex, with the effects of loss and gain of NF-Y function varying under different cellular and molecular contexts. Our experiments in pOPC revealed an increase in the transcription of NF-Yb following excitotoxic injury, yet, at the same time knockdown of NF-Yb decreased their viability, as did excitotoxic injury in Oli-neu.
Although appearing contradictory, these outcomes are consistent within the framework of NF-Y function discussed above, where roles in the regulation of both pro-and anti-apoptotic responses have been described (Gatta, Dolfini, & Mantovani, 2011). Importantly, the present data clearly show that dysregulation of NF-Yb consistently associates with reduced pOPC viability; therefore the direct modulation of NF-Y function is unlikely to provide a viable strategy for protecting OPC from excitotoxic injury.
Alterations in NF-Y function per se are likely to compromise OPC survial in vivo, therefore we performed a network analysis on gene expression microarrays to examine the transcriptional events associated with NF-Y in oligodendroglia. This analysis in Oli-neu subjected to excitotoxic injury and pharmacological disruption of NF-Y revealed that 43% of the genes commonly regulated in these models were associated with the regulation of apoptosis. On this basis these genes, and their downstream effectors, may provide promising targets for future work aiming to identify targets capable of protecting OPC from excitotoxic injury without compromising other essential NF-Y functions.

| Involvement of NF-Y in CNS dysfunction
As discussed above our data suggest an important role for NF-Y function in OPC survival. This conclusion is complemented by studies indicating NF-Y deregulation as a contributor to neurodegenerative processes in several polyglutamine (polyQ) expansion diseases (Huang, Ling, Yang, Li, & Li, 2011;Katsuno et al., 2010;Yamanaka et al., 2008).
In each case, disrupted NF-Y activity is associated with the accumulation of mutated polyQ-bearing proteins. Neurodegeneration then proceeds, in part, due to alterations in the transcription of NF-Y targets required for neuronal survival. This is exemplified in spinal and bulbar muscular atrophy, a polyQ disorder involving the accumulation of mutated androgen receptor (AR) protein in brain stem and spinal cord motorneurons. In this condition transcription of transforming growth factor-ß receptor type-II (TGFßR-II), a pro-survival NF-Y target, is impaired due to abnormal associations between NF-Y and mutated AR that probably prevent NF-Y from binding and activating TGFßR-II (Katsuno et al., 2010). Similarly, mutant Huntington, the pathological protein underlying Huntington's Disease (HD), forms inappropriate interactions with NF-Y leading to reduced transcription of heat shock protein 70 (HSP70), an NF-Y target expected to aid in the clearance of mutated proteins. These links between NF-Y dysfunction and CNS pathology and our present data suggesting anti-and pro-survival roles in oligodendroglia, highlight NF-Y dysfunction as an interesting target for evaluation in pathological conditions involving oligodendrocyte and myelin degeneration, including those mediated through excitotoxic injury (Groom et al., 2003;Kanwar et al., 2004;Pitt et al., 2000).
In conclusion, this work identifies NF-Yb as a key controller of Gria4 expression in OPC. AMPA-type glutamate receptors exert a powerful influence on the development and survival of OPC (Fannon et al., 2015;Gallo et al., 1996;Kougioumtzidou et al., 2017;Yuan et al., 1998), and are themselves under strong developmental regulation (Hossain et al., 2014;Itoh et al., 2002), thus further work exploring the interplay between NF-Y function and OPC differentiation represent a promising line of enquiry for studies aiming to uncover novel mechanisms regulating myelination. This work also highlights the NF-Y system as an important regulator of OPC survival during excitotoxic injury. BEGUM ET AL.

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OPC are exquisitely vulnerable to hypoxic-ischemic conditions (Back